大蒜精油丙烯基硫化物對人類皮膚癌細胞之抑癌活性及其分子機制探討

Abstract

皮膚癌是全球罹患率逐年增加的癌症之一，包括發生率最高的基底細胞癌（basal cell carcinoma; BCC）與死亡率較高的黑色素細胞癌（melanoma）。大蒜精油活性成分丙烯基硫化物（allyl sulfides）－二丙烯基單硫化物（diallyl sulfide; DAS）、二丙烯基二硫化物（diallyl disulfide; DADS） 與二丙烯基三硫化物（diallyl trisulfide; DATS）對多種癌細胞株具有抑制活性，但目前在人類皮膚癌細胞的研究卻很少見。本研究欲探討DAS、DADS與DATS對人類皮膚黑色素細胞癌A375細胞株，以及基底細胞癌BCC細胞株之抑癌活性及其分子機制。結果指出，丙烯基硫化物具有抑制A375與BCC細胞生長的作用，並以DATS的抑癌活性最高，可能與DATS可以促進細胞內活性氧物質（reactive oxygen species; ROS）的生成與鈣離子釋放有關，並導致細胞週期停滯於 G2/M期；而N-乙醯半胱胺酸（N-acetyl cysteine ; NAC）可以減低DATS對A375與BCC細胞生長的抑制作用。流式細胞儀及西方墨點法分析的結果發現，DATS可能透過氧化壓力造成DNA的損傷並活化p53路徑，調控G2/M期相關週期蛋白質的表現，並造成粒線體膜電位的下降與內質網壓力的生成，進一步啟動粒線體活化路徑，包括Caspase依賴型與非依賴型路徑，誘導A375與BCC細胞凋亡的發生。此外，DATS並不影響人類皮膚角質細胞HaCaT細胞株的存活率，顯示其對皮膚正常細胞和癌細胞之間具有選擇性抑制作用。在細胞轉移的體外實驗發現，DATS可能藉由調節黑色素細胞癌A375細胞Integrin與focal adhesion kinase (FAK) 的表現，降低基質金屬蛋白酶（matrix metalloproteinases; MMP）的活性，如MMP-2與MMP-9，而減低細胞移行（migration）、黏附（adhesion）、侵犯（invasion）之作用。以上結果證實，DATS抑制人類皮膚癌細胞生長的機制為誘導細胞週期停滯與細胞凋亡，並可能抑制黑色素細胞癌的轉移，因此推測DATS具有預防皮膚癌之潛力。

Global incidence rate of skin cancer is rapidly increased in recent years, including basal cell carcinoma (BCC) which is the most common type of skin cancer, and malignant melanoma is most metastatic tumor. Numerous studies have shown that diallyl sulfide (DAS), diallyl disulfide (DADS), and diallyl trisulfide (DATS), the active components of garlic essential oil, provide the anticancer activity in several cancer types, but their effects and related mechanisms of these allyl sulfides on skin cancer are unclear. The objective of this study was to investigate the anticancer activity and its molecular mechanisms of DAS, DADS, and DATS in human melanoma A375 cells and basal cell carcinoma BCC cells. We found that allyl sulfides decreased the cell viability of skin cancer cells, and DATS revealed better growth inhibition of A375 and BCC cells than DADS and DAS did. We further demonstrated that DATS increased intracellular reactive oxygen species (ROS) generation, induced cytosolic Ca(2+) mobilization, and decreased mitochondrial membrane potential. However, N-acetyl cysteine (NAC) attenuated the growth inhibition of A375 and BCC cells induced by DATS. Flow cytometry analysis and Western blot results showed the concordance for the expression of molecules involved in p53 pathway in response to DNA damage, G2/M arrest, ER stress, and apoptosis observed by cell cycle and cell viability analysis. Moreover, we detected the activation of Caspase-dependent and independent mitochondrial apoptosis pathway. DATS also displayed selective target of growth inhibition between skin cancer cells and normal keratinocyte HaCaT cells. Additionally, DATS inhibited cell migration, adhesion, and invasion of A375 cells by in vitro anti-metastatic assay. The anti-metastatic potency might relate to the regulation of Integrin and focal adhesion kinase (FAK), and the decrease in the activity of matrix metalloproteinases (MMPs) induced by DATS, including MMP-2 and MMP-9. In conclusion, DATS inhibited cell growth of human skin cancer cells cells via induction of cell cycle arrest and apoptosis, and might provid the anti-metastatic potency. These results suggest that DATS is a potential anticancer compound for skin cancer.